To develop a model system in which we could induce GCRs, we used the rare restriction enzyme I-SceI, whose 18 bp recognition sequence is not normally present in the human or mouse genome, to produce a single DNA DSB within a mammalian cell, based on the hypothesis that improper repair of these breaks could lead to GCRs. This enzyme has been used in a series of elegant studies to produce specific, non-random GCRs mediated by homologous recombination in mammalian cells. We generated a construct that expressed the Herpes simplex virus type I thymidine kinase (TK) gene under the control of the constitutive EF1a promoter, with the recognition sequence for the I-SceI restriction enzyme placed between the EF1a promoter and the TK gene. This pEF1aTK vector was introduced into the U937 cell line, and verified that expression of the TK gene conferred sensitivity to ganciclovir (GCV). We then carried out a series of experiments that utilized the negative selection provided by the expression of TK. Cells were transfected with an I-SceI expression vector and selected with GCV (to select for cells that had lost TK expression). We characterized over 100 independent clones. All of the clones had small deletions and showed evidence consistent with non-homologous end joining (NHEJ), such as microhomology or local sequence inversions. no clones showed evidence of having a GCR. We considered the possibility that the U937 cell line, which has a fairly normal karyotype [47 XX, t(10;11)] may not be an ideal choice for these types of experiments, and that a structurally unstable cell line might be more likely to produce GCR. The NCI 60 cell line panel has been assayed for numerical and structural instability;OVCAR8 showed the highest level of ongoing structural instability. We generated OVCAR-8 subclones that harbored a single copy of the pEF1aTK vector, and transfected these clones with I-SceI expression vectors. However, similar to the results with the U937 clones, all 31 GCV-resistant (GCVR) clones had small interstitial deletions and features of repair via NHEJ. We also considered the possibility that a potential disadvantage of the above approach is that it utilizes a negative selection system, and will generate clones that delete small (i.e. &gt;100bp) portions of the EF1a promoter or TK cDNA, leading to lack of TK expression and GCVR. Therefore, we developed a complementary vector that allowed positive selection. This vector contains a hygromycin phosphotransferase gene (HygroR;confers resistance to hygromycin) preceded by an I-SceI recognition sequence, but no promoter region. We transfected this promoter-less vector into OVCAR 8 cells, and identified clones that integrated a single copy. As anticipated, since the HygroR gene lacks a promoter, these cells are hygromycin sensitive. These cells were then transfected with an I-SceI expression vector, and selected with hygromycin, in the hopes of recovering rare clones that had undergone a GCR, and juxtaposed a promoter from a distant genomic region, thus allowing expression of the HygroR gene, leading to hygromycin resistance. All of the clones analyzed were vector capture events, in which a portion of SV40 regulatory sequences derived from the I-SceI expression vector have become juxtaposed to the hygroR gene. It may not be surprising that we were unable to generate GCRs by inducing a single DNA DSB, as other investigators have concluded that two induced breaks are required to produce a chromosomal translocation, and that the frequency of chromosomal translocations induced by a single DNA DSB in mouse embryonic stem (ES) cells is extraordinarily rare (&lt;5 x 10E08). However, an alternative, error prone NHEJ pathway has recently been described, and has been implicated in chromosomal translocations associated with malignancy. In addition, mice lacking one or more elements of the NHEJ repair pathway (such as Ku70/80, Lig4, or DNA-PKcs) or H2ax are prone to complex chromosomal rearrangements involving cellular proto-oncogenes. Therefore, we began studies to determine if inhibition of normal DNA DSB repair pathway components can lead to GCR after induction of a single DNA DSB. We obtained spontaneously immortalized H2ax-/- and Ku80 -/- murine fibroblast cell lines, and generated clones containing a single copy of the pEF1&amp;#945;TK vector. We transfected an I-SceI expression vector into H2ax-/- cells containing a single copy of the pEF1&amp;#945;TK vector (clone H2AX8), and selected GCV-resistant clones. The spectrum of DNA rearrangements identified in the H2ax-/- cells was markedly different than in any of the aforementioned studies. Of 26 clones characterized, only 2 were small interstitial deletions. 19 clones had vector capture events, and five clones represented GCRs, two balanced translocations and three chromosomal inversions of 3-78 Mb.